Distribution of catecholaminergic and peptidergic cells in the gerbil medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis with a focus on areas activated at ejaculation.

The posterodorsal preoptic nucleus (PdPN), lateral part of the posterodorsal medial amygdala (MeApd) and medial part of the medial preoptic nucleus (MPNm) are activated at ejaculation in male gerbils as assessed by Fos expression. We sought to immunocytochemically visualize substance P (SP), cholecystokinin (CCK), oxytocin, vasopressin and tyrosine hydroxylase (TH), a catecholaminergic marker, in the mating-activated cells, but the need for colchicine precluded behavioral testing. Instead, we detailed distributions of cells containing these molecules in the medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis (BST) and quantified their densities in the PdPN, MPNm and lateral MeApd for comparison to densities previously assessed for mating-activated efferents from these sites. TH cells were as dense in the PdPN and lateral MeApd as activated efferents to the anteroventral periventricular nucleus. In the lateral MeApd, TH cells were grouped where cells activated at ejaculation are clustered and where CCK cells form a ball. Lateral MeApd CCK cells and PdPN SP cells were as dense as activated efferents to the principal BST. Oxytocinergic PdPN cells and SP cells in the MPNm were as dense as mating-activated efferents to the lateral MeApd. If some oxytocin cells in the PdPN project to the neurohypophysis, as in rats, they could be a source of the oxytocin secreted at ejaculation. Since gerbils are monogamous and biparental, it was also interesting that, unlike monogamous prairie voles, they had few TH cells in the MeApd or dorsal BST, resembling promiscuous rats, hamsters and meadow voles.

Combined administration of the mixture of honokiol and magnolol and ginger oil evokes antidepressant-like synergism in rats.

Magnolia bark combined with ginger rhizome is a common drug pair in traditional Chinese prescriptions for the treatment of depression. In the present study, we examined antidepressant-like effects of the mixture of honokiol and magnolol (HMM) from magnolia bark and essential oil from ginger rhizome (OGR) alone and in combination in chronic unpredictable mild stress (CUMS) of rats. Behavioral (sucrose intake, immobility time of forced swimming test) and biochemical parameters [serotonin (5-HT) in prefrontal cortex, hippocampus, and striatum, gastric mucosa cholecystokinin (CCK) and serum gastrin (GAS) levels] were simultaneously examined in the CUMS rats. 20 mg/kg HMM alone, but not OGR, significantly increased sucrose intake and reduced immobility time in the CUMS rats. Moreover, 20 mg/kg HMM and 14 mg/kg OGR in combination exhibited significant synergistic effects on sucrose intake increase and immobility time reduction in the CUMS rats. HMM elevated 5-HT levels in various brain regions, and OGR reduced gastric mucosa CCK and serum GAS levels in the CUMS rats. These results suggested that the synergistic antidepressant-like effects of compatibility of HMM with OGR might be mediated simultaneously by regulation of the serotonergic and gastroenteric system functions. These findings also provided a pharmacological basis for the clinical application of this drug pair of magnolia bark and ginger rhizome in traditional Chinese medicine.

Species-specific modulation of pattern-generating circuits.

Phylogenetic comparison can reveal general principles governing the organization and neuromodulation of neural networks. Suitable models for such an approach are the pyloric and gastric motor networks of the crustacean stomatogastric ganglion (STG). These networks, which have been well studied in several species, are extensively modulated by projection neurons originating in higher-order ganglia. Several of these have been identified in different decapod species, including the paired modulatory proctolin neuron (MPN) in the crab Cancer borealis [Nusbaum & Marder (1989) J. Neurosci., 9,1501-1599; Nusbaum & Marder (1989), J. Neurosci., 9, 1600-1607] and the apparently equivalent neuron pair, called GABA (gamma-aminobutyric acid) neurons 1 and 2 (GN1/2), in the lobster Homarus gammarus [Cournil et al. (1990) J. Neurocytol., 19, 478-493]. The morphologies of MPN and GN1/2 are similar, and both exhibit GABA-immunolabelling. However, unlike MPN, GN1/2 does not contain the peptide transmitter proctolin. Instead, GN1/2, but not MPN, is immunoreactive for the neuropeptides related to cholecystokinin (CCK) and FLRFamide. Nonetheless, GN1/2 excitation of the lobster pyloric rhythm is similar to the proctolin-mediated excitation of the crab pyloric rhythm by MPN. In contrast, GN1/2 and MPN both use GABA but produce opposite effects on the gastric mill rhythm. While MPN stimulation produces a GABA-mediated suppression of the gastric rhythm [Blitz & Nusbaum (1999) J. Neurosci., 19, 6774-6783], GN1/2 activates or enhances gastric rhythmicity. These results highlight the care needed when generalizing neuronal organization and function across related species. Here we show that the 'same' neuron in different species does not contain the same neurotransmitter complement, nor does it exert all of the same effects on its postsynaptic targets. Conversely, a different transmitter phenotype is not necessarily associated with a qualitative change in the way that a modulatory neuron influences target network activity.

Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex.

Physiological and morphological properties of large non-pyramidal cells immunoreactive for cholecystokinin, parvalbumin or somatostatin were investigated in vitro in the frontal cortex of 18-22-day-old rats. These three peptides were expressed in separate populations including large cells. Cholecystokinin cells and parvalbumin cells made boutons apposed to other cell bodies, but differed in their firing patterns in response to depolarizing current pulses. Parvalbumin cells belonged to fast-spiking cells. Parvalbumin fast-spiking cells also included chandelier cells. In contrast, cholecystokinin cells were found to be regular-spiking non-pyramidal cells or burst-spiking non-pyramidal cells with bursting activity from hyperpolarized potentials (two or more spikes on slow depolarizing humps). Large somatostatin cells belonged to the regular-spiking non-pyramidal category and featured wide or ascending axonal arbors (wide arbor cells and Martinotti cells) which did not seem to be apposed to the somata so frequently as large cholecystokinin and parvalbumin cells. For electron microscopic observations, another population of eight immunohistochemically-uncharacterized non-pyramidal cells were selected: (i) five fast spiking cells including one chandelier cell which are supposed to contain parvalbumin, and (ii) three large regular-spiking non-pyramidal cells with terminals apposed to somata, which are not considered to include somatostatin cells, but some of which may belong to cholecystokinin cells. The fast-spiking cells other than a chandelier cell and the large regular-spiking non-pyramidal cells made GABA-positive synapses on somata (4% and 12% of the synapses in two small to medium fast-spiking cells, 22% and 35% of the synapses in two large fast-spiking cells, and 10%, 18% and 37% of the synapses in three large regular-spiking non-pyramidal cells). A few terminals of the fast-spiking and regular-spiking non-pyramidal cells innervated GABAergic cells. About 30% of the fast-spiking cell terminals innervated spines, but few of the regular-spiking non-pyramidal cell terminals did. A fast-spiking chandelier cell made GABA-positive synapses on GABA-negative axon initial segments. These results suggest that large GABAergic cells are heterogeneous in neuroactive substances, firing patterns and synaptic connections, and that cortical cells receive heterogeneous GABAergic somatic inputs.

Galanin and cholecystokinin in cultured magnocellular neurons isolated from adult rat supraoptic nuclei: a correlative light and scanning electron microscopical study.

Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.

Cholecystokinin effect on human lymphocyte ionized calcium and mitogenesis.

Cholecystokinin (CCK) is a peptide present in large amounts in gut, brain, and neurons innervating lymphatic tissues. Plasma CCK levels increase in enterally alimented patients. Enteral alimentation is also associated with enhanced immune function. The effects of CCK and a CCK antagonist were studied on human peripheral blood mononuclear cells (H-PBMC), lymphocyte intracellular ionized calcium ([Ca2+]i), and lymphocyte mitogenesis. CCK receptors transduce their signal via the release of [Ca2+]i. CCK octapeptide caused a specific increase in [Ca2+]i measured by Fura-2 fluorometry in H-PBMC and human T helper lymphocytes. Neither gastrin-17 nor pentagastrin produced a signal. While the highly specific CCK antagonist MK329 blocked the CCK [Ca2+]i signal, it had no effect on the PHA-mediated signal. At high dosages (10(-7)-10(-8) M), CCK was a comitogen with "complete" lymphocyte mitogens such as anti-CD3 monoclonal antibody (mAb) or low-dose PHA, but not for "partial" mitogens such as phorbol esters. CCK comitogenic effect occurred even in the presence of cyclosporine. CCK radioimmunoassay demonstrated that H-PBMC contained CCK and that anti-CD3 mAb- or PHA-mediated H-PBMC mitogenesis caused release of CCK. MK329 blocked PHA and anti-CD3 mAb mitogenesis and CCK comitogenic effects. We conclude that CCK octapeptide may be a coregulator of lymphocyte Ca2+ activation signals. The immunologically beneficial effect of enteral nutrition may, in part, be mediated by increased levels of CCK.

Partial purification, tissue distribution and modulatory activity of a crustacean cholecystokinin-like peptide.

Reversed-phase chromatography was used to separate several forms of cholecystokinin-like peptides (CCKLP) from the pericardial organs (PCOs) of the spiny lobster Panulirus interruptus. Fast protein liquid chromatography of PCOs, stomatogastric ganglia (STGs) and eyestalks revealed five peaks of CCKLP (peaks A-E) that were common to all three tissues, as well as two additional peaks (peaks F and G) in the STG. Peaks A-E were present in the hemolymph of fed, but not starved, lobsters. The bioactivity of peaks A-E was tested on the gastric mill rhythm of the isolated STG. Only peak E elicited activity. The effects of peak E included activating the gastric mill rhythm in quiescent preparations and strengthening existing rhythms in a dose-dependent manner. Further purification of peak E by high performance liquid chromatography resolved this peak into two immunoreactive peaks, one of which retained its bioactivity. The effects of peak E were blocked by the CCK antagonist proglumide. These results are consistent with a role for peak E in the feeding-induced activation of the gastric mill.

The relationship of calbindin-containing neurons with substance P, Leu-enkephalin and cholecystokinin fibres: an immunohistochemical study in the rat thalamus.

In the rat thalamus, immunoreactivity for the calcium binding protein calbindin (Cb) is mostly confined to neuronal cell bodies, sometimes revealing proximal dendrites, of the midline, intralaminar and posterior regions. Substance P (SP)-, cholecystokinin (CCK)- and Leu-enkephalin (L-ENK)-immunoreactive (ir) elements in the thalamus are fibre-like structures, intermingled with punctate elements probably representing axonal arborizations and their synaptic boutons. These peptidergic fibres are unevenly distributed in several thalamic domains, including the areas that contain Cb-ir neurons. The relationship between Cb-ir cell bodies and these three different peptidergic systems of thalamic innervation was studied with immunohistochemistry. Single-labelling experiments on adjacent sections and double immunostaining on the same section were performed. A considerable overlap between Cb-ir perikarya and SP-ir fibres was found in most thalamic nuclei. In particular, in the intralaminar nuclei and posterior complex, SP-ir punctate elements were frequently observed in close proximity to Cb-ir cell bodies and dendrites. On the other hand, no consistent topographical correspondence between Cb-ir perikarya and CCK- or L-ENK-ir fibres was evident. Altogether, the present data suggest a selective anatomical and, possibly, functional relationship between SP and Cb in at least a subpopulation of rat thalamic neurons.

The regional distribution of thyrotropin releasing hormone, leu-enkephalin, met-enkephalin, substance P, somatostatin and cholecystokinin in the rat brain and pituitary.

There was no apparent difference in the regional distribution of neuropeptides in the brain of male and female rats. The highest levels of immunoreactive leu-enkephalin, TRH, substance P and somatostatin were found in the hypothalamus, while the striatum and the cerebral cortex had the highest concentrations of met-enkephalin and cholecystokinin respectively. The lowest concentrations of these were found in the cerebellum. Enkephalins (cerebral cortex), substance P (cerebral cortex and brain stem), and somatostatin (brain stem and striatum) showed higher level in the female while enkephalin and substance P contents in the anterior pituitary were higher in the male.

Distribution of cholecystokinin-like-immunoreactive neurons in the guinea pig forebrain.

The distribution of cholecystokinin (CCK)-immunoreactive nerve fibers and cell bodies was studied in the forebrain of control and colchicine-treated guinea pigs by using an antiserum directed against the carboxyterminus of CCK octapeptide (CCK-8) in the indirect immunoperoxidase technique. Virtually all forebrain areas examined contained immunoreactive nerve fibers. A dense innervation was visualized in; neocortical layers II-III, piriform cortex, the medial amygdala, the medial preoptic area, a circumventricular organ-like structure located at the top of the third ventricle in the preoptic area, the subfornical organ, the posterior bed nucleus of the stria terminalis, the posterior globus pallidus (containing labeled woolly fiber-like profiles), the ventromedial hypothalamus, the median eminence, and the premammillary nucleus. A moderately dense innervation was visualized elsewhere excepted in the septum and thalamus where labeled axons were comparatively few. Immunoreactive perikarya were abundant in: neocortex (especially layers II-III), piriform cortex, amygdala, the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic paraventricular (parvicellular part), arcuate, and dorsomedial (pars compacta) nuclei, the dorsal and perifornical hypothalamic areas, and throughout the thalamus. Areas also containing a moderate number of labeled cell bodies were the medial preoptic area, the globus pallidus, the caudate-putamen, and the periventromedial area in the hypothalamus. Immunostained perikarya were absent or only occasionally observed in the septum, the suprachiasmatic nucleus, the magnocellular hypothalamoneurohypophyseal nuclei, and the ventral mesencephalon. In the adenohypophysis, corticomelanotrophs were labeled in both males and females, and thyrotrophs were labeled in females only. This distribution pattern of CCK-8 immunoreactivity is compared to those previously recorded in other mammals. This shows that very few features are peculiar to the the guinea pig. It is discussed whether some interspecific differences in immunostaining are real rather than methodological.

Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: II. Bed nucleus of the stria terminalis and amygdala.

The distribution of cholecystokinin-immunoreactive (CCK-I) cell bodies was studied in the bed nucleus of the stria terminalis (BST) and amygdaloid complex of colchicine-treated male and female rats. Immunoreactive cells were visualized in the BST medial amygdaloid (MeA), central lateral, basolateral, basolateral ventral, medial, intercalated, anterior cortical, and posterior cortical nuclei and the amygdalohippocampal zone. Several significant sex differences were observed. In the male, a dense aggregation of CCK-I cell bodies was visualized in the MeA, especially in the dorsocaudal part and in the encapsulated part of the BST. In comparison, female rats had relatively fewer immunoreactive cells in both of these regions. In the lateral and basolateral amygdaloid nuclei, however, more CCK-I cells were visualized in the female than in the male, but the difference was not statistically significant. These data provide characterization of a sexually differentiated CCK system. In addition, we observed that the number of CCK-I cells in the BST and posterodorsal part of the MeA was substantially reduced after castration. The number of CCK-I cells in female rats, however, was not significantly reduced after ovariectomy in any of the regions studied. These findings imply that the steroid regulation of CCK is sexually differentiated. The sexually dimorphic distribution of CCK-I cells in areas that are targets of steroid hormones and regulate reproductive processes is consistent with the possibility that CCK participates in central integration of sensory and steroidal input that modulates reproductive behavior.

Evidence for an absence of estrogen-concentration by CCK-immunoreactive neurons in the hypothalamus of the female rat.

It is becoming increasingly clear that the neuropeptide cholecystokinin (CCK), widely distributed in the rat hypothalamus and limbic system, is subject to both organizational and activational influences of steroid hormones. Sex differences in numbers of CCK-immunoreactive elements have been demonstrated in sexually dimorphic structures such as the bed nucleus of the stria terminalis, medial preoptic nucleus, and ventromedial nucleus of the hypothalamus. Steroid activation of CCK has been indicated by findings that hypothalamic CCK levels and binding capacity vary over the estrous cycle. These studies, in combination with evidence of CCK mediation of sexually differentiated functions, prompted us to test for estrogen concentration among CCK-containing cells of the female rat hypothalamus by combining the techniques of immunohistochemistry and autoradiography. A method employing 2-week ovariectomies and perfusion fixation with 4% paraformaldehyde was compatible with the localization of both estrogen-accumulating and CCK-immunoreactive cell bodies. The maintenance of numbers of CCK-positive cells after gonadectomy suggested that expression of this peptide may not be directly regulated by ovarian steroids in female rats. This suggestion was substantiated by the finding that, with rare exceptions, CCK-immunoreactive cells did not concentrate estrogen in tissues collected from the anterior-posterior extent of the bed nucleus of the stria terminalis, medial preoptic nucleus, anterior hypothalamic area, and paraventricular nucleus.

Immunohistochemical distribution of cholecystokinin, dynorphin A and Met-enkephalin neurons in sheep hypothalamus.

The distribution of cholecystokinin (CCK)-, Met-enkephalin (M-ENK)- and dynorphin (DYN)-like immunoreactive perikarya were examined in the sheep hypothalamus using the peroxidase-anti-peroxidase technique. CCK- and DYN-containing neurons were found primarily in the suprachiasmatic nucleus (SCH) and supraoptic nucleus (SO). No CCK- or DYN-containing neurons were found in the paraventricular nucleus (PVN). M-ENK-containing neurons were found mainly in the PVN of the hypothalamus. In addition, M-ENK neurons were found in the dorsomedial (DMH), lateral (LH), anterior (AH) and periventricular hypothalamic areas. The distribution of these neuropeptides may provide a basis for understanding differences in responsiveness to centrally administered peptides.

Measurement and characterization of neuronal cholecystokinin using a novel radioreceptor assay.

This study describes a novel radioreceptor assay (RRA) for cholecystokinin (CCK) which is the first to measure and characterize brain CCK using a technique not dependent on the generation of peptide antibodies. The CCK RRA utilizes the mouse cerebral cortex CCK receptor as the binding source and [125I]BH-CCK-8 as the radiolabelled probe. [125I]BH-CCK-8 bound to the central CCK receptor with a Kd of 1.82 nM and a Bmax of 1.21 pmol/g tissue. Unlabelled CCK-8 displaced the specific binding of [125I]BH-CCK-8 with an inhibition constant of 3.84 nM. CCK was extracted (90% methanol) from discrete brain regions (mouse) and quantified using the CCK RRA. The amygdala contained the highest concentration of CCK (394 +/- 21 pmol/g tissue), followed by the olfactory bulbs (306 +/- 19 pmol/g tissue) and cerebral cortex (298 +/- 21 pmol/g tissue). Moderate levels of CCK were found in the hippocampus (212 +/- 18 pmol/g tissue), striatum (146 +/- 15 pmol/g tissue) and hypothalamus (129 +/- 9 pmol/g tissue). Low levels of CCK were recorded in the pons (45 +/- 5 pmol/g tissue), medulla (41 +/- 3 pmol/g tissue) and spinal cord (29 +/- 3 pmol/g tissue), whilst no CCK was detected in the cerebellum. The molecular forms of CCK in amygdala, cerebral cortex and hypothalamus were characterized using RRA in conjunction with HPLC. CCK-8 was identified as the major molecular form (88%, 94% and 91% of total CCK activity in amygdala, cortex and hypothalamus, respectively) with a smaller component attributable to CCK-4 (8%, 5% and 6% of the total CCK activity).

An intragastric meal releases the putative satiety factor cholecystokinin from hypothalamic neurons in cats.

The release of cholecystokinin-like immunoreactivity (CCK-LI) from feline hypothalamus was studied in relation to a meal by use of the push-pull perfusion technique. While levels of CCK-LI in the perfusate of overnight-fasted anesthetized cats were below assay sensitivity (less than 7 pg/30 min), intragastric administration of a carbohydrate-amino acid meal elicited a 3-fold increase in CCK-LI, identified by high-performance liquid chromatography as the C-terminal octapeptide of CCK (CCK-8). Examination of an extrahypothalamic site showed no comparable release in CCK-LI. Intravenous infusions of CCK-8 at doses imitating physiological blood levels up to 400 times higher than those seen after physiological stimulation demonstrated the existence of a CCK blood-brain barrier in the lateral hypothalamus. These observations provide support that CCK may play a physiological role in termination of feeding behavior.

Increase in hypothalamic cholecystokinin following acute and chronic morphine.

Recent evidence supports an antagonistic interaction between cholecystokinin (CCK) and opiate peptides. The present study determined the effects of various levels of morphine treatment on hypothalamic levels of CCK as determined by radioimmunoassay. Acute treatment with morphine sulfate (10 mg/kg) or implantation of one morphine pellet (75 mg free base) increased levels of CCK in whole hypothalamus. Increased exposure to morphine by either chronic injections or implantation of two pellets did not result in a further change in whole hypothalamic CCK levels. In samples dissected into hypothalamic subregions, the effect of morphine on CCK levels was localized to medial but not lateral or posterior regions. These experiments extend earlier in vitro findings and suggest that some of the physiological and behavioral effects of opiate peptides may result from modulation of endogenous CCK.

Cholecystokinin and substance P concentrations in discrete areas of the rat brain: sex differences.

Cholecystokinin (CCK) and substance P (SP) concentrations were measured in discrete brain areas of adult male and diestrous female rats. Significant sex differences in CCK concentration were found in the ventromedial hypothalamic area, medial and lateral preoptic area, nucleus of the diagonal band of Broca, ventral tegmental area, entorhinal and in several cortical areas. No sex differences in SP concentrations were observed in any of these areas. However, significant sex differences in SP concentration were found in the amygdala. These data indicate that the CCK and to some extent the SP systems are sexually differentiated in certain brain areas.

Coexistence of neuropeptides in projection neurons of the thalamus in the cat.

Coexistence of neuropeptides was suggested by double-staining immunohistochemistry in projection neurons in the thalamus of the cat; cholecystokinin (CCK)-like immunoreactivity (LI) and vasoactive intestinal polypeptide (VIP)-LI in the rostral group of the intralaminar nuclei, CCK-LI and neurotensin (NT)-LI in the anterodorsal nucleus and NT-LI and VIP-LI in the laterodorsal nucleus.